[重组腺相关病毒载体介导的RNA干扰抑制爱泼斯坦-巴尔病毒编码的潜伏膜蛋白1对鼻咽癌细胞体内转移能力的影响]

[Influence of suppression of Epstein-Barr Virus-encoded latent membrane protein 1 by rAAV vector mediated RNA interference on metastatic ability of nasopharyngeal cancer cells in vivo].

作者信息

Liu Xiong, Li Gang, Zhang Bao, Wang Lu, Li Xiao-hua, Li Xiang-ping

机构信息

Department of Otolaryngology, Nanfang Hospital, Nanfang Medical University, Guangzhou 510515, China.

出版信息

Zhonghua Zhong Liu Za Zhi. 2009 May;31(5):324-9.

PMID:19799078

Abstract

OBJECTIVE

To study the effect of Epstein-Barr virus (EBV) encoded latent membrane protein 1 (LMP-1) on the tumor growth and metastasis of nasopharyngeal carcinoma (NPC) cells in vivo and its possible mechanism.

METHODS

To construct two recombinant adeno-associated virus (rAAV): rAAV-shRNA-LMP-1 and rAAV-EGFP (enhanced green fluorescent protein). Multiplicity of infection (MOI) was confirmed by using different titre of rAAV-EGFP to transfect a NPC cell line, C666-1. Then C666-1 cells were transfected by rAAV-shRNA-LMP-1 at MOI titre and the inhibiting efficiency of target gene's expression was confirmed by reverse transcription polymerase chain reaction (RT-PCR). C666-1 cells treated by RNAi on LMP-1 in vitro was directly inoculated into the liver via laparotomy under direct vision to establish the animal model of NPC xenograft in liver and lung metastasis from the liver. The biological effect and its mechanism after "gene silencing" of LMP-1 on NPC cells tumorigenesis and metastasis were observed by the primary intrahepatic tumor formation and lung metastasis and the expression of matrix metalloproteinases-9 (MMP-9) revealed by immunohistochemistry.

RESULTS

The transfection efficiency was higher than 95% with 5 x 10(4) virus genome (v.g)/cell with rAAV-EGFP. The expression of target gene was inhibited more than 90%, assessed by RT-PCR after transfection with rAAV-shRNA-LMP-1 at a dose of 5 x 10(4) v.g/cell. The primary tumor volume implanted in the liver of rAAV-shRNA-LMP-1 treatment was (0.2527 +/- 0.1152) cm3, with no significant difference in comparrison with rAAV-EGFP control group [(0.2533 +/- 0.0754) cm3, P>0.05]. But the rate of intrahepatic tumor formation was 50.0% and the rate of lung metastasis was 33.3% of the rAAV-shRNA-LMP-1 group, significantly lower than those in the rAAV-EGFP group (P<0.05). The survival time (15.50 +/- 2.47) d of rAAV-shRNA-LMP-1 group was significantly longer than that in the rAAV-EGFP group [(11.50 +/- 1.22) d, P<0.05]. Immunohistochemistry indicated suppression of LMP-1 by rAAV-shRNA-LMP-1 can down-regulate the expression of MMP-9 significantly.

CONCLUSION

The expression of LMP-1 can be suppressed effectively by rAAV mediated RNA interference. The suppression of LMP-1 expression has no effect on cell growth but can inhibit the metastasis in vivo, probably through down-regulating the expression of MMP-9.

摘要

目的

研究爱泼斯坦-巴尔病毒（EBV）编码的潜伏膜蛋白1（LMP-1）对鼻咽癌（NPC）细胞在体内肿瘤生长和转移的影响及其可能机制。

方法

构建两种重组腺相关病毒（rAAV）：rAAV-shRNA-LMP-1和rAAV-增强绿色荧光蛋白（EGFP）。通过使用不同滴度的rAAV-EGFP转染NPC细胞系C666-1来确定感染复数（MOI）。然后以MOI滴度用rAAV-shRNA-LMP-1转染C666-1细胞，并通过逆转录聚合酶链反应（RT-PCR）确认靶基因表达的抑制效率。体外经RNA干扰LMP-1处理的C666-1细胞通过直视下剖腹术直接接种到肝脏中，以建立NPC肝移植瘤及肝肺转移的动物模型。通过原发性肝内肿瘤形成和肺转移以及免疫组织化学检测基质金属蛋白酶-9（MMP-9）的表达，观察LMP-1“基因沉默”后对NPC细胞肿瘤发生和转移的生物学效应及其机制。

结果

rAAV-EGFP以5×10⁴病毒基因组（v.g）/细胞转染效率高于95%。用剂量为5×10⁴ v.g/细胞的rAAV-shRNA-LMP-1转染后，通过RT-PCR评估，靶基因表达抑制超过90%。rAAV-shRNA-LMP-1处理组植入肝脏的原发性肿瘤体积为（0.2527±0.1152）cm³，与rAAV-EGFP对照组[（0.2533±0.0754）cm³，P>0.05]相比无显著差异。但rAAV-shRNA-LMP-1组肝内肿瘤形成率为50.0%，肺转移率为33.3%，显著低于rAAV-EGFP组（P<0.05）。rAAV-shRNA-LMP-1组的生存时间（15.50±2.47）d显著长于rAAV-EGFP组[（11.50±1.22）d，P<0.05]。免疫组织化学表明，rAAV-shRNA-LMP-1对LMP-1的抑制可显著下调MMP-9的表达。